Project Summary Understanding the molecular mechanisms that drive a pluripotent progenitor cell to differentiate into a specific cell type is germane to Developmental Biology and has important significance in regenerative medicine. Past studies have illustrated that dynamic cardiac transcription programs (CTPs) guide cardiogenesis during development and support cardiac structure and function in homeostasis. While the contributions of key cardiac-specific transcription factors and cardiogenic signaling pathways have been clearly delineated, molecular mechanisms that coordinate the expression of cardiogenic genes to drive myocardial cell specification and direct the maturation of cardiomyocytes remain elusive. Our recent studies using both zebrafish and mouse models indicate that the multi-functional protein Rtf1 is a transcription regulator that orchestrates cardiac gene programs responsible for myocardial specification and differentiation. Knockdown of rtf1 in mouse embryonic stem cells inhibits the cardiac gene program and prevents cardiac differentiation. In vivo, Rtf1 deficient zebrafish and mouse embryos lack myocardial progenitor cells and cannot develop a heart. We also found that Rtf1 deficiency in committed cardiomyocytes impairs cardiac gene program. Collectively, these findings demonstrate a need for Rtf1 activity in myocardial cells at multiple developmental stages. Insights into how Rtf1 regulates dynamic CTPs come from our structure-function analysis showing differential requirements for Rtf1's Plus3 and HMD domains in two temporally distinct cardiogenic events; the Plus3 domain is required for the activation of the CTP and myocardial specification whereas the HMD domain influences histone modifications and directs heart tube morphogenesis. These exciting findings lead to our overarching hypothesis that Rtf1 controls dynamic transcriptional programs in myocardial cells during development by CTP activation and epigenetic modulation. We will employ a set of new transgenic zebrafish lines to define the transcriptional networks of Rtf1 in myocardial progenitors and committed cardiomyocytes. We will investigate how Rtf1's Plus3 domain coordinates the expression of cardiogenic genes to drive the multi-potent mesodermal cells to a myocardial fate (Aim1). We will also interrogate the hypothesis that the Rtf1's HMD domain controls heart tube morphogenesis by influencing the propagation and/or maturation of newly differentiated cardiomyocytes via epigenetic modulation (Aim 2). Successful completion of the proposed projects will provide new mechanistic insights into the transcriptional regulation of myocardial specification and differentiation and will pave the way for the development of novel therapeutic strategies to treat heart diseases.